Synthetic cytokine circuits that drive T cell infiltration into immune-excluded tumors

CAR T cells are ineffective against solid tumors with immunosuppressive microenvironments. To overcome suppression, we engineered circuits in which tumor-specific synNotch receptors locally induce production of the inflammatory cytokine, interleukin-2 (IL-2). These cytokine delivery circuits can potently enhance CAR T cell infiltration and clearance of immune-excluded tumors (immunocompetent models of pancreatic cancer and melanoma) without systemic toxicity. The most effective IL-2 induction circuit acts in an autocrine and TCR/CAR-independent manner, bypassing suppression by host cells that either consume IL-2 or inhibit TCR signaling. These engineered autocrine cells are able to establish an effective foothold in the tumors, likely because synNotch-induced IL-2 production can cooperatively enable initiation of CAR-mediated T cell expansion and killing. Thus, it is possible to reconstitute synthetic T cell circuits that activate the outputs ultimately required for a robust anti-tumor response, but in a manner that evades key points of tumor suppression.


Materials and Methods
Viral DNA constructs Primary human T cells were engineered with constructs cloned into a second generation 5' selfinactivating lentiviral backbone (pHR). All lentiviral constructs and sequences are detailed in Tables S1 and S2. Lentiviral synthetic cytokine circuits cells were made by transducing human T cells with a synNotch component and a response element component. SynNotch genes were expressed constitutively from mouse PGK promoters whereas response elements were controlled by a 5xGAL4 repeat with a minimal CMV promoter. The payloads controlled by synNotch were either expressed as "cytokine IRES mCherry" or "BFP P2A cytokine," where the cytokine was super-2 (sIL-2), human IL-2, IL-7, or IL-15. For in vitro experiments, bystander T cells were transduced with an SFFV eGFP vector. For in vivo experiments, the autocrine circuit was generated by placing the synNotch and response element in one lentiviral vector and either a SFFV effluc P2A mCherry or PGK BFP P2A anti-NY-ESO-1 TCR in the other vector. Paracrine circuits were created by placing the synNotch and response element in one cell and the effluc or NY-ESO-1 TCR in the other cell. Primary mouse T cells were engineered with constructs cloned into pMIG2 alternatively known as pMSCV or the self-inactivating pRetroX plasmid (Takara Bio). All retroviral constructs and sequences are detailed in Tables S3 -S6. Primary immune cell culture Primary human CD8 + and CD4 + T cells and NK cells were isolated from leukapheresis packs using EasySep kits (Stemcell Technologies), following which they were frozen in RPMI with 20% human AB serum and 10% DMSO. For assays, frozen T cells were thawed in human T cell media (hTCM; X-VIVO media [Lonza], 5% human AB serum, 10 mM n-acetyl cysteine, 55 µM β mercaptoethanol) with IL-2 (always 30 U/mL unless otherwise specified) and resuspended at 1e6 cells/mL. One day after thawing T cells were activated with 25 μL anti-CD3/anti-CD28 coated beads (Dynabeads Human T-Activator CD3/CD28 [Gibco]) per 1e6 T cells. 24 hours after activation T cells were infected by incubating 1e6 activated T cells with 1 to 1.5 mL of lentivirus for 24 hours. Following infection viral supernatant was removed from cells which were resuspended in media with IL-2. Cells were sorted for positive transduction on a FACSAria Fusion 5 days after activation based on expression of a fluorescent protein marker or for positive staining of a Myc-tag (anti-Myc-tag antibody, 9B11, Alexa Fluor 647 conjugate, Cell Signaling Technology, Cat# 2233) on synNotch, or both. Cells were then expanded by counting daily and diluting with hTCM with IL-2 to a cell concentration of 5e5 cells/mL for an additional week to allow cells to rest from initial activation prior to in vitro analysis or in vivo use. Frozen NK cells were thawed and resuspended in hTCM 24 hours prior to use.
Mouse T cells were isolated from spleens and lymph nodes of female C57/Bl6 or OT-1 mice, which were mechanically dissociated over a 40 micron filter. RBCs were lysed using RBC lysis buffer (Biolegend) prior to negative selection for CD3 + or CD8 + T cells (StemCell) with purity confirmed post-sort by surface staining. Mouse T cells were grown in RPMI supplemented with 10% fetal bovine serum, 2 mM Glutamax, 20 mM HEPES, 1% pen/strep, 1 mM sodium pyruvate, 0.05 mM beta-mercaptoethanol, and 50 IU/mL human IL-2. Mouse T cells were activated on day of isolation with either anti-mouse CD3/CD28 dynabeads (ThermoFisher) or OVA peptide (GenScript). 24 hours after activation 1e6 mouse T cells were spinfected at 2000g for 2 hours at 32C on retronectin coated (15 ug/mL, Takara Bio) non-TC coated 24 well plates with 1 to 1.5 mL of retrovirus and 4 ng/mL polybrene (Sigma-Aldrich). Retrovirus was removed after 4 hours and mouse T cells expanded until 3 days after activation when they were sorted on a FACSaria as above. Mouse T cells were then expanded daily by counting and diluting with mTCM + IL-2 to maintain a concentration of 1e6 cells/mL for 9 days after activation prior to use with in vitro or in vivo assays.

Virus Production
Lentivirus was produced using Lx293t lentiviral packaging cells (Takara bio, Cat# 632180) that were seeded in 6-well plates at 7e5 cells/well and 24 hours later transfected with pHR constructs and pCMV and pMD2.g packaging plasmids using FuGene HD (Promega) following manufacturer's protocol. 48 hours after transfection viral supernatant was collected and filtered prior to use with human T cell cultures. Retrovirus was producing using Plat-E retroviral packaging cells (Cell Biolabs, Cat# RV-101) that were seeded in 6 well plates at 9e5 cells/well and 24 hours later transfected with pMIG2 or pRetroX constructs using FuGene HD (Promega) following manufacturer's protocol. 48 hours after transfection viral supernatant was collected and filtered prior to use with mouse T cell cultures.
In-Vitro Assays T cells and target cells were washed of residual media and cytokines by two rounds of centrifugations at 400xg for 4 minutes followed by resuspension in hTCM without IL-2. In some cases, T cells were stained with 1:5000 CellTrace CFSE proliferation stain (Molecular Probes) following manufacturer's protocol. Magnetic anti-HA or anti-Myc-tag antibody-coated beads (Pierce) were washed 3 times with hTCM using a magnet before using. Immune cells were seeded at 2.5e5 cells/mL and K562 cells at 1.25e5 cells/ml. IL-2 was added as indicated. For beads, 2.5 μL (in terms of original suspension before washing) per well was used. Every 2 days, wells were triturated and 50 μL cell suspension was taken to analyze by flow cytometry. A 96well magnet array was used to retain magnetic beads. The media was then replenished: cells were pelleted, 100 μL of old media was removed, then 150 μL fresh hTCM was added to restore the volume to 200 μL.

Mouse Experiments
Prior to injection into mice, T cells were washed in PBS, resuspended at 10 times the injection amount per mL, and 100 μL was injected via the tail vein on day 0. Mouse T cells were administered to female 6 to 12 week old C57/Bl6 mice (Jackson Labs Strain #000664) and human T cells were administered to female 6 to 12 week old NSG (NOD-scid IL2Rgamma null ) mice. Heterotopic K562, A375 and KPC tumors were prepared by washing cells in PBS three times, resuspending at 10 times the injection amount per mL, and injecting 100 μL subcutaneously in each flank on day 0 (K562 tumors), day -4 (A375 tumors) or day -7 to -9 (KPC tumors). Orthotopic B16-F10 tumors were prepared as before but resuspended at 25 times the injection amount per mL, and 25 uL was injected intradermally on day -7 to -9. Orthotopic KPC tumors were prepared as before with 125,000 cells implanted into the tail of the pancreas in 25 uL in a 1:1 mix with Matrigel (Fisher Scientific). Sub-cutaneous and intra-dermal tumor size was measured by caliper while orthotopic tumor size was measured by luciferase signal. Bioluminescence imaging was performed using an IVIS Spectrum (Perkin Elmer). Mice were injected intraperitoneally with 200 μL of 15 mg/mL d-luciferin (Goldbio) and imaged 15 minutes later. Typical sample size per group was 5 mice with randomization at time of tumor implantation. Tumor measurements were performed by staff blinded to treatment groups, only mice that were engrafted tumors were included in analysis. All animal studies were performed under the UCSF Office of Research Institutional Animal Care and Use Program approval number AN183960-02N.
In-Vitro Immune Cell Assays Flow cytometry was performed either on a BD LSRii or BD Fortessa X-20 with high-throughput system. Analysis of flow data was performed in FlowJo (FlowJo, LLC). For counting, constant volumes were taken from each well and all events were counted within those volumes. IL-2 production in primary human T cells was measured in 96 well flat bottom plates with A375 cells in co-culture with T cells. After 24 hours T cells were exposed to golgi-plug/stop (BD Biosciences) for four hours and then fixed and stained with PE-Texas Red anti-CD4 (Biolegened, Cat# 317448, AB_2565847) and BUV-737 conjugated anti-human IL-2 (BD Bioscience, Cat# 564446) before flowing. ELISA measurements of IL-2 production from mouse T cells were performed using a human or mouse IL-2 quantikine ELISA kit (R&D systems, Cat# M2000). Absolute T Cell proliferation was measured by adding 2 uL of anti-myc Tag beads to T cells in a 200 uL volume on Day 0 and sampling 50 uL of cells with replacement of media every 2 days. Viable T Cell counts were quantified using 1:500 Sytox (Molecular Probes) in PBS and accounting for dilution of wells. T cell proliferation was also analyzed by dilution of CFSE cell trace dye (Thermo Fisher). NK cell expansion was measured in co-culture of T cells, NK cells and K562 cells. Cells were stained with 1:100 anti-CD3 and 1:100 anti-CD8 fluorescent conjugated antibodies in flow buffer (PBS, 5% FBS) for 30 minutes at 4°C followed by 1:500 DRAQ7 (Molecular Probes) in flow buffer. Mouse T cell cytotoxicity was tested against GFP labeled target cells that were plated at indicated count in 100 uL in 96 well flat bottom plates and placed in an Incucyte Live-Cell Imaging System (Sartorius). T cells were added at indicated count in 100 uL of mTCM without IL2 and survival of GFP labeled target cells was recorded by imaging. Mouse T cell differentiation measured with BV605 anti-PD-1 (Biolegend, Cat# 135220, AB_2562616) and PE Lag3 (Biolegend, Cat #125208, AB_2133343). Human T cell differentiation measured with FITC anti-CD450RO (Biolegend, Cat #304242, AB_314420) and Analysis of tumor specimens: IHC Tumors samples were collected for IHC and fixed in 10% formalin for 24 to 48 hours prior to preservation in 70% ETOH. Tissue was embedded in paraffin, sectioned and mounted for staining with single chromogenic anti-CD3 (clone SP7) antibody at the UCSF Biorepository core facility.
Analysis of tumor specimens: CyTOF (Cytometry by time of flight) Tumor samples for CyTOF were processed immediately after collection. Tumor samples were minced and digested in RPMI 1640 with 1 mg/ml collagenase IV and 0.1 mg/ml DNase I for 30 min at 37°C with shaking. Digested tumor samples were filtered with a 70 µm cell strainer and washed with PBS + 5mM EDTA at 4°C. Cells were resuspended 1:1 with PBS + 5mM EDTA + 50μM Cisplatin (Sigma) for exactly 60s prior to quenching 1:1 with PBS + 5mM EDTA + 0.5% BSA to determine viability. Tumor sample was fixed for 10 min at room temperature using 1.6% PFA and frozen at -80°C. Mass-tag cellular barcoding and antibody staining of samples for CyTOF were performed as previously described (1). A summary of all antibodies used for CyTOF are detailed in Tables S7. All antibodies were conjugated at the UCSF Parnassus Flow Cytometry Core. Each antibody clone and lot was titrated to optimal staining concentration using primary murine samples. CyTOF was performed on a Helios mass cytometer (Fluidigm) with data analysis performed in CellEngine and R.   Table S3. Name (all in pMIG2 backbone) Order of Elements (:: indicates same transcript) anti-Meso CAR huPGK promoter, Kozak sequence, CD8 signal sequence::V5-tag::anti-mesothelinnanobody::cd8hinge:cd8transmembrane::41bb::cd3zeta BFP-p2a-mIL2 huPGK promoter, Kozak sequence, BFP::p2a::mIL2 Constituent components of pMIG2 lentiviral vectors